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Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors—palbociclib, ribociclib, or abemaciclib—immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms. Clinical CDK4/6 inhibitors are used and tested to treat a variety of cancer types. Here, the authors identify that these drugs work in two ways, a known catalytic role to inhibit kinase activity and a newly discovered noncatalytic role to displace CDK inhibitor p21 from CDK4 but not CDK6 complexes.

A crystal structure of the active form of cyclin-dependent kinase 4 (CDK4) provides insight into regulation of the cell cycle and the mechanism of action of a drug used for breast cancer therapy. The protein p27 has been thought to act as a CDK inhibitor. Guiley et al. performed a structural analysis of active complexes of CDK4 with cyclin D1 (CycD1) and p27 (see the Perspective by Sherr). The results showed that p27 actually remodels the active site of CDK4 to allow full activation when p27 is phosphorylated on tyrosine (phosp27). Furthermore, they found that the breast cancer drug palbociclib, a CDK4 inhibitor, doesn't actually interact with active phosp27-CDK4-CycD1 trimers. Instead, it appears that the drug, which shows promise in the clinic, binds to inactive CDK4 monomers and prevents interaction with p27. Science , this issue p. eaaw2106 ; see also p. 1315 Crystal structures clarify the regulation mechanism of a kinase complex linked to cancer. The p27 protein is a canonical negative regulator of cell proliferation and acts primarily by inhibiting cyclin-dependent kinases (CDKs). Under some circumstances, p27 is associated with active CDK4, but no mechanism for activation has been described. We found that p27, when phosphorylated by tyrosine kinases, allosterically activated CDK4 in complex with cyclin D1 (CDK4-CycD1). Structural and biochemical data revealed that binding of phosphorylated p27 (phosp27) to CDK4 altered the kinase adenosine triphosphate site to promote phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and other substrates. Surprisingly, purified and endogenous phosp27-CDK4-CycD1 complexes were insensitive to the CDK4-targeting drug palbociclib. Palbociclib instead primarily targeted monomeric CDK4 and CDK6 (CDK4/6) in breast tumor cells. Our data characterize phosp27-CDK4-CycD1 as an active Rb kinase that is refractory to clinically relevant CDK4/6 inhibitors.

In women with hormone-receptor–positive metastatic breast cancer that had progressed after endocrine therapy, palbociclib plus fulvestrant was associated with progression-free survival of more than 9 months, as compared with less than 4 months with fulvestrant alone. Approximately 80% of breast cancers express estrogen receptors, progesterone receptors, or both. Endocrine therapies are the mainstay of treatment for these hormone-receptor–positive cancers, substantially reducing the relapse rate after presentation with early-stage cancer. 1 Despite advances in endocrine therapy, many women have a relapse during or after completing adjuvant therapy. The care of these women remains a considerable clinical challenge. Single-agent treatment with an aromatase inhibitor or tamoxifen has shown limited clinical benefit. 2 , 3 The selective estrogen-receptor degrader fulvestrant has modest activity in this population of patients, 4 , 5 and the development of effective therapies that can reverse resistance to endocrine therapy . . .

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5–MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5–MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.

Concerted multidisciplinary efforts have led to the development of Cyclin-Dependent Kinase inhibitors (CDKi’s) as small molecule drugs and chemical probes of intracellular CDK function. However, conflicting data has been reported on the inhibitory potency of CDKi’s and a systematic characterization of affinity and selectivity against intracellular CDKs is lacking. We have developed a panel of cell-permeable energy transfer probes to quantify target occupancy for all 21 human CDKs in live cells, and present a comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of 46 clinically-advanced CDKi’s and tool molecules. We observed unexpected intracellular activity profiles for a number of CDKi’s, offering avenues for repurposing of highly potent molecules as probes for previously unreported targets. Overall, we provide a broadly applicable method for evaluating the selectivity of CDK inhibitors in living cells, and present a refined set of tool molecules to study CDK function. Cyclin-dependent kinase (CDK) inhibitors are widely used both in the clinic and for basic research aimed at dissecting the specific cellular functions of specific CDKs. Here, the authors report the development of a panel of fluorescent reporter probes and provide a comprehensive profile of the inhibitory activity of several CDK inhibitors towards all 21 CDKs in living cells.

Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on induction of the p19 Arf –p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumour-suppressive senescence response in mice and cells devoid of Skp2 . Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19 Arf –p53 pathway nor DNA damage, but instead depend on Atf4, p27 and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19 Arf –p53 response is impaired, whereas a Skp2–SCF complex inhibitor can trigger cellular senescence in p53/Pten-deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof-of-principle evidence that pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy. Senescence kills tumours Recent studies suggest that cellular senescence — an irreversible form of cell-cycle arrest — can halt tumour growth in vitro . Hui-Kuan Lin et al . now identify a previously unknown pathway that drives senescence without the involvement of most of the known mediators of senescence. Instead, it signals via the transcription factor Atf6, and the cyclin-dependent kinase inhibitors p27 and p21. The pathway is uncovered by inactivation of the proto-oncogene Skp2 , but only in the context of oncogenic signalling. Targeting the Skp2 complex pharmacologically restricts tumorigenesis by inducing cellular senescence, suggesting that such drugs may be effective in cancer prevention and therapy. Cellular senescence — an irreversible cell-cycle arrest — has been implicated in suppressing tumour formation or growth. A new cellular signalling pathway that drives senescence has now been identified. This pathway does not involve most known mediators of senescence, and instead signals via the proteins Atf4, p27 and p21. Inactivating the proto-oncogene Skp2 in the context of oncogenic signalling can induce senescence through this new pathway, indicating that drugs that target Skp2 might be useful in cancer treatment.

Mechanisms underlying the generation of induced pluripotent stem cells (iPSC) and keeping iPSC stability remain to be further defined. Accumulated evidences showed that iPSC reprogramming may be controlled by the cell-division-rate-dependent model. Here we reported effects of absence of mouse p27 or p18 on iPSC generation efficiency and genomic stability. Expression levels of cyclin-dependent kinases inhibitors (CDKIs), p21, p27, and p18 decreased during iPSC reprogramming. Like p21 loss, p27 or p18 deficiency significantly promoted efficiency of iPSC generation, whereas ectopic expression of p27, p18, or treatment with CDK2 or CDK4 inhibitors repressed the reprogramming rate, suggesting that CDKIs-regulated iPSC reprogramming is directly related with their functions as CDK inhibitors. However, unlike p21 deletion, absence of p27 or p18 did not increase DNA damage or chromosomal aberrations during iPSC reprogramming and at iPSC stage. Our data not only support that cell cycle regulation is critical for iPSC reprogramming, but also reveal the distinction of CDKIs in somatic cell reprogramming.

The mammalian cell cycle is precisely controlled by cyclin-dependent kinases (CDKs) and related pathways such as the RB and p53 pathways. Recent research on long non-coding RNAs (lncRNAs) indicates that many lncRNAs are involved in the regulation of critical cell cycle regulators such as the cyclins, CDKs, CDK inhibitors, pRB, and p53. These lncRNAs act as epigenetic regulators, transcription factor regulators, post-transcription regulators, and protein scaffolds. These cell cycle-regulated lncRNAs mainly control cellular levels of cell cycle regulators via various mechanisms, and may provide diversity and reliability to the general cell cycle. Interestingly, several lncRNAs are induced by DNA damage and participate in cell cycle arrest or induction of apoptosis as DNA damage responses. Therefore, deregulations of these cell cycle regulatory lncRNAs may be involved in tumorigenesis, and they are novel candidate molecular targets for cancer therapy and diagnosis.

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle 1 , 2 , when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein 3 – 5 . Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell’s progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling. We uncover the mechanism underlying the restriction point phenomenon, suggest a role for cyclin-dependent kinase 4 and 6 activity in S and G2 phases, and explain the behaviour of cells following loss of mitogen signalling.

Key Points Many cell cycle proteins are overexpressed or overactive in human cancers, in particular, D-type and E-type cyclins, cyclin-dependent kinases (CDK4, CDK6 and CDK2), Polo-like kinase 1 (PLK1) and Aurora kinases (Aurora A and Aurora B). In transgenic mice, overexpression of several of these cell cycle proteins induces or contributes to tumorigenesis, revealing their prominent oncogenic roles. Some of these cell cycle proteins are also required for tumorigenesis, and their ablation in mice impairs tumour formation induced by specific genetic lesions or by carcinogen treatment, as demonstrated for several cyclins (D1, D2 and D3) and CDKs (CDK4, CDK6, CDK2 and CDK1), as well as for checkpoint kinase 1 (CHK1). Importantly, in some cases the continued presence of a cell cycle protein has also been shown to be required for tumour maintenance and progression, for example, for cyclin D1, cyclin D3 and CDK4, thereby providing a clear rationale for targeting these proteins in cancer treatment. Kinases involved in cell cycle checkpoint function such as CHK1 and WEE1 also constitute potential therapeutic targets. Their inhibition compromises checkpoint function, causes excessive DNA damage and eventually leads to apoptosis, particularly in cells with compromised p53 function. CDK4/6-selective inhibitors, such as palbociclib, ribociclib and abemaciclib, have shown significant benefits in clinical studies, particularly in breast cancer, but also in non-small-cell lung cancer, melanoma and head and neck squamous cell carcinoma. Importantly, following demonstration of a substantial improvement in progression-free survival, combination of palbociclib and letrozole received accelerated approval for first-line treatment of patients with advanced ER + HER2 − breast cancer. Inhibitors of PLK1, such as rigosertib and volasertib, have also shown encouraging results in clinical phase II/III studies for patients with myelodysplastic syndromes and acute myelogenous leukaemia, respectively, and several phase III trials are currently ongoing. Compounds targeting Aurora A, particularly alisertib, have been extensively studied in preclinical models and demonstrated synergy with many other targeted therapies, leading to tumour regression in various cancer models. Moreover, clinical studies revealed encouraging activity of alisertib in peripheral T cell lymphoma, non-Hodgkin lymphoma, non-small-cell lung cancer and breast cancer. Proteins regulating cell cycle progression are involved in the formation of most cancer types. This Review discusses the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment, results of clinical trials, as well as future therapeutic potential of various cell cycle inhibitors. Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.